The long-term goal of this project remains to determine the molecular and cellular mechanisms by which testosterone regulates spermatogenesis. Testosterone is essential for fertility and targets Sertoli cells in the mammalian testis through the androgen receptor (AR) to produce factors and provide an environment required for germ cell survival and development. Numerous critical Sertoli cell activities have been found to be dependent on testosterone including 1) the formation of tight junctions between Sertoli cells that form the essential blood-testis barrier, 2) the remodeling of Sertoli cell-round spermatid attachments and the retention of round spermatids, 3) the release of mature spermatozoa. Although testosterone had been known to be required for these functions, the mechanisms by which testosterone acts are not well understood. We recently characterized an alternative, rapid (<1 min) and sustained mechanism of testosterone action (the non-classical pathway) that causes the phosphorylation and activation of the Src and ERK kinases, the epidermal growth factor receptor and the CREB transcription factor. We will test the overall hypothesis that critical spermatogenesis supporting functions of testosterone are mediated via the non-classical pathway. Aim 1 is to determine whether non-classical signaling by testosterone regulates Sertoli-Sertoli and Sertoli-germ cell adhesion in culture. We will test the hypothesis that non-classical testosterone actions are required for Sertoli-germ cell attachment. We will determine whether the non-classical pathway regulates mRNA expression, protein levels or the phosphorylation status of factors known to maintain Sertoli-Sertoli adhesion and Sertoli-germ cell attachment. Aim 2 is to determine whether non-classical testosterone actions are required to release mature spermatozoa from seminiferous tubules. We will determine whether testosterone activates Src and ERK kinases in cultured seminiferous tubules. We will quantify spermatozoa release from cultured seminiferous tubule fragments in the presence and absence of testosterone and inhibitors of the non-classical testosterone signaling pathway. This study will determine mechanisms by which testosterone supports spermatogenesis and identify new drug targets that may be used to regulate male fertility.